Complex suitable for carrying out a method of purifying pre-S hepatitis B surface antigen

ABSTRACT

There is disclosed a complex comprised of an insoluble polymer carrier to which monomeric human albumin is covalently bound and of a pre-S hepatitis B surface antigen bound in an elutable form to the monomeric human albumin by its pre-S(2)- and/or pre-S(1)-region. This complex may be used for therapeutic and diagnostic purposes and enables the rapid and efficient purification of pre-S-HBsAg by affinity chromatography.

This application is a divisional of Ser. No. 07/578,939, filed Sep. 7,1990, now abandoned.

The invention relates to a complex comprised of an insoluble polymercarrier to which human albumin is covalently bound and of a pre-Shepatitis B surface antigen (pre-S-HBsAg) as well as to a method for thepurification of pre-S-HBsAg.

From EP-A2-0 243 103, a method of purifying pre-S-HBsAg is known, whichconsists in disrupting yeast cells expressing recombinant pre-S-HBsAgand separating pre-S-HBsAg from the cell contents by affinitychromatography. Polymerized human serum albumin (polyalbumin) covalentlybound to a matrix serves as the adsorbent for pre-S-HBsAg. Thispolyalbumin is a product synthetically prepared in a high-molecular formby cross-linking agents, e.g., glutaraldehyde. The pre-S-HBsAg isadsorbed on the polyalbumin by its pre-S(2)-region and, after havingwashed off interfering substances, is eluted from the matrix andsubjected to a secondary purification step.

It was evidenced that an efficient polyalbumin receptor existed in thepre-S region of HBsAg, which is composed of a polypeptide including 55amino acids and encoded by a section of the hepatitis virus DNAimmediately preceding the S-region ("pre-S2") (Valenzuela et al.,Bio/Technology Vol. 3: 317-320, 1985). Furthermore, it was demonstratedthat both this immediately preceding region (pre-S2) and the totalpre-S-region, in connection with the S-region (pre-S1), encode surfaceproteins that constitute receptors to hepatitis B virus (HBV) for thebinding to cell membranes of liver cells.

HBsAg that contains a pre-S region for the first time was obtained frominfected human plasma in 1979 (Neurath and Strick, Arch. Virol., 60:79-81, 1979) and later on was produced also by way of geneticengineering by Valenzuela et al. (Bio/Technology 3: 317, 1985, andNature 311: 67, 1984) and Paoletti et al. (PNAS 81: 193, 1984).

Subsequently, various recombinantly expressed pre-S-containing surfaceantigens were suggested as vaccines to induce the formation ofantibodies against HBV. These antibodies are directed against thereceptor of the virus, thus preventing its binding to the liver cell andthe infection involved.

The purification of the antigens by affinity chromatography throughpolyalbumin has the disadvantage that impurities, yet even toxicsubstances, such as glutaraldehyde, are introduced into the eluate dueto the use of cross-linking agents in the production of polymerizationproducts.

It is the object of the present invention to eliminate this difficultyand to provide a complex which, in addition to the opportunity of arapid and efficient affinity chromatographic purification ofpre-S-HBsAg, offers new applications for therapeutic and diagnosticpurposes.

The complex according to the invention is comprised of an insolublepolymer carrier based, in particular, on agarose or dextrane, to whichmonomeric human albumin is covalently bound, and of a pre-S hepatitis Bsurface antigen bound in an elutable form to the monomeric human albuminby its pre-S(2)- and/or pre-S(1)-region.

It was found according to the invention that pre-S-HBsAg would becomplexed to the monomeric human albumin to an extent sufficient foraffinity chromatographic purification only if the latter were covalentlybound to the carrier. If the albumin merely binds by way of adsorption,the pre-S-HBsAg can be complexed to the albumin not at all or onlyextremely slightly such that an affinity chromatographic purificationwill not be possible. Another advantage of the complex according to theinvention consists in that pre-S-HBsAg may be readily eluted, theprotein thus being recoverable in a more gentle manner and a greateryield.

An insoluble polymer carrier to which monomeric human albumin iscovalently bound is required for the formation of the complex of theinvention. This insoluble polymer carrier, thus, also falls within thescope of the invention. To produce the insoluble polymer carrieraccording to the invention, any polymer capable of covalently bindingproteins upon suitable activation may be used. The following carriersubstances may be used:

organic polymers, such as polyamides and vinyl polymers (polyacrylamide,polystyrene and polyvinyl alcohols and derivatives thereof), as well as

natural polymers, such as cellulose, dextrane, agarose, chitin andpolyamino acids, and

inorganic polymers, such as silica gel, glass and metal hydroxides.

These carrier substances may be used in the form of particles, e.g., asmolecular sieves, in the form of membranes or of plates, e.g., asmicrotiter plates.

Preferably, the insoluble polymer carrier is based on agarose ordextrane.

The complex according to the invention is storable over extended periodsof time, i.e., both in the aqueous phase in which the carrier is presentin the swollen state (e.g., as an affinity resin) and in the lyophilizedstate (e.g., as a membrane or a microtiter plate). Lyophilization,preferably, is carried out in a volatile buffer containing glycine orglucose.

Pre-S-HBsAg can be isolated from the complex according to the inventionin high purity. The invention also relates to the use of the complexaccording to the invention for the preparation of diagnostics andvaccines, the vaccines being applicable both for active immunizationagainst hepatitis B and for obtaining specific immunoglobulin of donorsimmunized with such vaccines.

The carrier according to the invention may be loaded with pre-Shepatitis B surface antigen in a simple manner by contacting an aqueoussolution of hepatitis B surface antigen with the carrier, thepre-S-containing fractions of the hepatitis surface antigens beingselectively adsorbed on the albumin molecules.

The method according to the invention for the purification of pre-Shepatitis B surface antigen by using a complex according to theinvention is characterized in that

the complex is washed with a buffer solution to remove possibly presentimpurities, and

the pre-S hepatitis B surface antigen selectively adsorbed on themonomeric human albumin is cleaved and recovered either by treating thecomplex with an eluting agent containing chaotropic substances, such asurea, guanidine hydrochloride, thiocyanate, potassium chloride,magnesium chloride or potassium iodide; or by treating the complex withdetergents; or by treating the complex with a pH modifying agent;whereupon a secondary purification step is carried out, if necessary.

A preferred embodiment of the method according to the invention ischaracterized in that ionic detergents, in particular sodiumdesoxycholate and taurodesoxycholic acid, or zwitterionic detergents, inparticular 3[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonateand 3[(3-cholamidopropyl)-dimethyl-ammonio]-2-hydroxy-1-propanesulfonate, or non-ionic detergents, in particular octylglucopyranosides, may be used as the detergents.

It has proved that the quarternary structure of the eluted pre-S-HBsAgis not affected when using these detergents.

Preferably, the secondary purification step after elution comprises gelfiltration, ultracentrifugation, hydrophobic chromatography, lectinaffinity chromatography or ion exchange chromatography. Any of thesepurification steps yields pre-S-HBsAg in a purity greater than 90%.

The invention will be explained in more detail in the following:

The pre-S-HBsAg can be expressed from recombinant vaccinia virus thatcontains the genetic information of the pre-S-HBsAg. This technique hasbeen described in the literature (Moss et al., Nature 311: 67 (1984)).

At first, a high-titer virus stock of recombinant vaccinia virus wasprepared by infecting vero cells with 1 PFU/cell. After 2 to 3 days ofincubation at 37° C. the infected cells are shaken into the medium andpelleted by centrifugation at 5,000 g for 20 min. The supernatant ispoured off and stored at 4° C. The cells are washed three times in PBS,whereupon a trypsin solution is added to the cell suspension until afinal concentration of trypsin of 0.025% by mass is reached. After this,the suspension is gently stirred at 37° C. for 30 min, pooled with thesupernatant, aliquoted into ampoules and deepfrozen at -80° C. By thismethod, the virus titer is increased by approximately ten times ascompared to that usually obtained in cell medium.

A vero cell inoculum is prepared by passaging cells in plastic Roux andRoller bottles (Nunc) in order to produce sufficient cells to be able toinoculate a 6-liter fermenter, which, in turn, serves as the inoculumfor a 40-liter vessel. To this end, a single ampoule of vero cells of adefined passage number at first is thawed and passaged to provide 12confluent Roller bottles.

The cells are trypsinized, resuspended in medium 199 with 5% by mass offoetal calf serum and mixed with a suspension of microcarriers (Cytodex3, Pharmacia) and pumped into the fermenter to give a finalconcentration of 2×10⁸ cells and 5 g microcarrier per liter. At thisstage, additional medium is added in an amount of one third of the finalworking volume.

The cells are allowed to adhere to the microcarriers for a period ofthree hours while slowly stirring the suspension. After this, furthermedium (DMEM containing 5% by mass of fetal calf serum) is added inorder to obtain the final working volume. As soon as a cell density of 6to 8×10⁸ /liter has been achieved, a continuous perfusion with DMEM(containing 5% by mass of fetal calf serum) is started. Upon achievementof a cell density of approximately 5×10⁹ /liter, the microcarriers aretrypsinized and the cells with the microcarriers are pumped into the40-liter fermenter, which contains additional 5 g microcarrier perliter. Upon adsorption, the fermenter is filled to a volume of 40 litersand cultivation proceeds as described above.

When a cell density of 5×10⁹ /liter has been reached, the medium ispumped off after the microcarriers have settled. Five liters of themedium containing the recombinant microorganism are pumped into thefermenter so as to give a m.o.i.-value of about 2 PFU per recombinantcell. After adsorption of the virus, the fermenter is filled with medium199 (containing 5% by mass of fetal calf serum) to a volume of 40 litersand is perfused with 40 liters of the same medium for 40 hours. Afterthis period of time, about 80% of the cells has detached from themicrocarriers and is pumped off together with the medium.

The remaining cells still adhering to the microcarriers are detachedtherefrom by washing with medium under rapid stirring and are pumped offand pooled with the first fraction. The microcarriers are removed bymeans of a 70 um sieve. By centrifugation in a Beckmann JFC-Z continuousflow rotor, the cells are pelletized at 16,000 g. The medium isconcentrated by ultrafiltration in the Pellikon system(Millipore-Waters). Urea is added to the concentrated medium until an 8Msolution adjusts. This solution is dialyzed.

The concentrated vero cell supernatant is added to a column packed withan insoluble polymer carrier substance to which human serum albumin iscovalently bound. The coupling method is known to depend on the chemicalnature of the carrier. When using Sepharose, activation is effected withCNBr, for instance, whereupon the monomeric human serum albumin iscoupled this activated Sepharose.

After washing of the column with buffer 1 consisting of 0.2M sodiumacetate (pH 4.0) and 0.5M NaCl, with buffer 2 consisting of 0.1M Tris(pH 8.0) and 0.5M NaCl, with a solution 3 consisting of 8M urea, andfinally with buffer 3 consisting of 0.02M Tris (pH 7.4), the vero cellsupernatant is applied to the column. As soon as the supernatant hasbeen adsorbed by the column, the non-adsorbed proteins are washed fromthe column with buffer 4 consisting of 0.02M Tris (pH 7.4) and 0.5MNaCl. The adsorbed pre-S(2)-HBsAg is eluted with sodium thiocyanate inbuffer 4 (1 to about 4M, preferably 3M) at a pH of from 6 to about 8,preferably at pH 7 (buffer 5) or with 8M urea in buffer 4, preferablywith 4M urea (buffer 6). This separation according to the invention withmonomeric human albumin results in a 145-fold enrichment of pre-S-HBsAg.Its identity is revealed by the immunoblotting technique (Burnette,Anal. Biochem., 112, 195, 1981) and by silver stained polyacrylamidegels (silver staining; Morrissey, Anal. Biochem., 117, 307, 1981), thepurity amounting to at least 80%.

The pre-S(2)-HBsAg may be further purified, i.e., by gel filtration onSepharose (Pharmacia). In doing so, the fraction obtained above isdialyzed against a buffer consisting of 0.02M Tris (pH 7) and applied tothe Sepharose column activated with 0.02M Tris (pH 7). The purity of thepre-S-HBsAg can be shown to be greater than 90% by silver staining andimmunoblotting.

The realization of the method according to the invention is furtherillustrated by the following exemplary embodiments.

EXAMPLE 1 Purification of pre-S(2)-HBsAg by monomeric human albuminaffinity chromatography

The recombinant vaccinia virus with the generic information for thepre-S(2)-HBsAg was obtained using the techniques described by Moss etal. (Nature 311: 67, 1984). The high-titer stock of the recombinantvaccinia virus was prepared as described above. The vero cells forinfection with the recombinant virus were grown under the sameconditions as described above. The concentrated dialyzed medium with thepre-S(2)-HBsAg was obtained in the previously described manner.

Monomeric human albumin was covalently bound to CNBr-activated Sepharose4B (Pharmacia) following the manufacturer's instructions. After this, acolumn was packed with a volume of 50 ml matrix and washed with 500 mlof buffer solution 1,500 ml of buffer solution 2,500 ml of solution 3and, finally, 500 ml buffer solution 4.

The clarified vero cell supernatant contained 8500 mg protein and 95 mgpre-S(2)-HBsAg and was pumped onto the prepared column at a flow rate of100 ml/hour. When the total amount had entered the column, thenon-adsorbed proteins were washed from the column with buffer solution4. The pre-S(2)-HBsAg was eluted with 3M sodium thiocyanate, pH 7(buffer solution 5). Identification and purity analysis were effected bysilver staining and immunoblotting following polyacrylamide gelelectrophoresis.

Duplicate aliquots of the fractions eluted from the column wereincubated for 15 min at 100° C. in a buffer consisting of 2% by mass ofsodium dodecyl sulfate (SDS), 0.125M Tris-HCl (pH 6.8) and 100 mMdithiotreitol. The samples were electrophoresed through 12.5% by masspolyacrylamide separating gel for 2.5 hours at 65 mA/gel (Laemmli,Nature 227: 680, 1971). One set of the gel samples was stained withsilver nitrate to visualize the polypeptides. The other set of thesamples was assayed by immunoblotting, using rabbit antiserum.

The eluted fractions contained 42 mg protein and 68 mg pre-S(2)-HBsAg.Thus, a 145-fold enrichment of the pre-S(2)-HBsAg had been achieved.

The following Examples 2 and 3 illustrate the secondary purification ofthe fractions obtained in Example 1.

EXAMPLE 2

A pre-S(2)-HBsAg-containing solution obtained according to Example 1 wasfurther purified by column chromatography on Sepharose 4B (Pharmacia).At first, the column was prepared and washed with 500 ml 0.02M Tris (pH7.0) at a flow rate of 30 ml/hour. The pre-S(2)-HBsAg-containingsolution was dialyzed against this buffer, and the dialyzed antigenpresent at a concentration of 10 mg/12 ml in addition to 6.2 mgprotein/12 ml was added to the column. The column was activated with0.02M Tris (pH 7.0) and the eluted pre-S(2)-HBsAg-containing fractioncontained 4.8 mg protein and 8.2 mg pre-S(2)-HBsAg. Silver-staining andimmunoblotting of the polyacrylamide gels carried out in the mannerdescribed in Example 1 revealed a purity of the pre-S(2)-HBsAg greaterthan 90%.

EXAMPLE 3

A pre-S(2)-HBsAg-containing solution obtained according to Example 1 wasfurther purified by column chromatography on lentil-lectin Sepharose 4B(Pharmacia). At first, the column was prepared and washed with 100 ml0.02M Tris (pH 7.0) at a flow rate of 50 ml/hour. Thepre-S(2)-HBsAg-containing solution was dialyzed against this buffer, andthe dialyzed antigen present at a concentration of 21 mg/25 ml inaddition to 13 mg protein/25 ml was applied onto the column at a flowrate of 25 ml/hour. After this, the non-adsorbed material was washed offwith Tris buffer solution and the pre-S(2)-HBsAg was eluted with Trisbuffer solution containing 5% by mass of alphamethylmannoside. Theeluate contained 9.1 mg protein and 15.6 mg pre-S(2)-HBsAg. Silverstaining and immunoblotting of the polyacrylamide gels were carried outas described in Example 1 and revealed a purity of the pre-S(2)-HBsAggreater than 90%.

The purification of hepatitis B surface antigens bearing thepre-S(1)-region instead of the pre-S(2)-region or pre-S(2)- andpre-S(1)-regions is accomplished in a manner analogous to the exemplaryembodiments described. The method according to the invention may,indeed, be used to purify any protein that carries a pre-S(1)- and/or apre-S(2)-region of HBsAg.

The following Examples 4 to 6 describe the use of various carriersubstances and forms as well as coupling methods that serve the requiredpurpose of use.

EXAMPLE 4 Coupling of monomeric human albumin to silicate carriers

Possible carriers are silica gels or glass microbeads (controlled poreglass beads=CPG).

10 g of an aminated glass carrier (aminopropyl CPG, Pierce) are shakenwith 100 ml of a 2.5% aqueous glutaraldehyde solution for four hours atroom temperature. Upon activation, the carrier is thoroughly washed freefrom aldehyde with deionized water. The thus activated carrier isincubated with 200 mg human serum albumin in 0.1M phosphate buffer (pH8.0) for one hour. After blocking with 1M ethanolamine and thoroughwashing with phosphate buffer, the carrier is ready for use.

200 ml of a pre-S(2)-HBsAg-containing cell culture supernatant (11 mgpre-S(2)-HBsAg, 950 mg protein) are incubated with 10 ml of the affinitycarrier for two hours at 14° C. Unbound protein is removed by washingwith a buffer on a sintering suction filter and the specifically boundpre-S(2)-HBsAg is eluted by incubation of the packed carrier in 10 ml 8Murea solution. This assay yielded 6.8 mg highly pure pre-S(2)-HBsAg atan overall protein amount of 5.4 mg (determination according toBradford).

EXAMPLE 5 Coupling of monomeric human albumin to membranes

Membranes, e.g., of nylon, polyvinylidene difluoride or cellulosepolyacrylamide mixed polymers are possible carriers. Nylon membrane(e.g., Zetabind, CUNO) is partially hydrolyzed by incubation in HCl/H₂O. The released amino groups are reacted with 1M oxaldialdehyde in thepresence of 0.1M sodium cyanoborohydride at pH 7 for two hours. Afterwashing with deionized water, human serum albumin (10 mg/ml in 0.1Mphosphate buffer pH 7) is coupled to this membrane activated withreactive aldehyde groups in the presence of 0.1M sodiumcyanoborohydride. Excess reactive groups are blocked by the addition of1M ethanolamine. After washing with phosphate buffer (0.1M; pH 8), themembrane may be used to selectively bind pre-S(2)-containing proteins.

The binding capacity of a thus produced membrane is 5 to 10 ugpre-S(2)-HBsAg/cm².

EXAMPLE 6 Coupling of a monomeric human albumin to microtiter plates

To this end, functionalized polystyrene plates, such as, e.g.,Amino-Plate (including primary amino groups) or Carbo-Plate (includingcarboxyl groups) (Nissho Iwai Corp., Tokyo) or similar materials may beused.

200 ul of a solution of N-ethoxycarbonyl-2-ethoxy-1,2-dihydro-quinoline(40 mM in 50% aqueous ethanol) are pipetted into each well of a 96-wellmicrotiter plate and incubated for two hours at 40° C. After carefulwashing with ethanol and deionized water, 200 ul of a 1% aqueoussolution of human serum albumin are each pipetted into a well andincubated at +4° C. over night. After blocking with 1M ethanolamine or1M acetate buffer pH 4 and subsequent washing, the plate is ready foruse.

100 ul of a pre-S(2)-HBsAg-containing cell culture supernatant arepipetted into the prepared microtiter plate in increasing dilutions andincubated at +4° C. for two hours. After washing with buffer andincubation with enzyme-labeled anti-pre-S(2)-HBsAg (monoclonalantibodies) following the known ELISA technique, the bound portion ofpre-S(2)-protein is determined by substrate addition according to knownmethods of absorption spectroscopy.

If the microtiter plate is loaded with SH-denaturized pre-S(2)-HBsAg orsynthetic pre-S(2)-peptide, a thus treated plate may be used forassaying anti-pre-S(2)-antibodies (e.g., from patients' sera).

10 mg pre-S(2)-HBsAg in 10 ml phosphate buffer (0.1M; pH 7.0) areincubated with 100 myl mercaptoethanol in 10% sodium dodecyl sulfate(SDS) for 5 min at 100° C. In doing so, the immunologically activeS-antigen of HBsAg is destroyed (Milich D. R. et al., EnhancedImmunogenicity of the Pre-S-Region of Hepatitis B Surface Antigen,Science 227: 1195-1199 (1985)). After blocking of the SH-groups with 500mg iodoacetamide for one hour, excess reaction products are eliminatedby dialysis and the pre-S(2)-antigen is diluted to 100 ul protein/mlwith phosphate buffer pH 7.4.

A prepared HSA microtiter plate is coated with this pre-S(2) solutionand subsequently washed. Thus prepared plates may be stored even in thedry state.

100 ul of an anti-pre-S(2)-containing rabbit serum are each pipettedinto the wells in increasing dilutions. After incubation at +4° C. overnight, bound pre-S(2)-antibodies are assayed by means of absorptionspectroscopy by incubation with enzyme-labeled anti-rabbit serum andsubsequent substrate addition.

EXAMPLE 7 Binding capacity and yield of pre-S(2)-HBsAg when usingvarious albumin carrier complexes

Human serum albumin (HSA) and polymerized human serum albumin (poly-HSA)were covalently and adsorptively bound to various carrier substances incomparable amounts. The binding capacity and the yield of pre-S(2)-HBsAgafter elution were determined by means of a HBsAg-specific ELISA methodand are summarized in the following Table:

    ______________________________________                                                                Capac-                                                                        ity   % yield after elution with                                     Type of  mg/g  2M   4M   8M   3M                               Albumin                                                                              Carrier bond     gel   U    U    U    KSCN                             ______________________________________                                        mono-  CNBr-   covalent 7.6   45   85   60   50                               meric  Sepha-  via                                                                   rose    NH.sub.2                                                       mono-  anti-   adsorp-  0     --   --   --   --                               meric  HSA     tive                                                                  Sepha-                                                                        rose                                                                   poly-  anti-   adsorp-  5.8   --   --   --   --                               meric  HSA     tive                                                                  Sepha-                                                                        rose                                                                   poly-  CNBr-   covalent 6.4   12   40   55   45                               meric  Sepha-  via                                                                   rose    NH.sub.2                                                       ______________________________________                                         U = Urea                                                                 

It is apparent from the Table that higher yields of pre-S(2)-HBsAg areobtained with the insoluble polymer carrier according to the invention,with which the monomeric human albumin is covalently bound to Sepharosein the present exemplary embodiment, than with Sepharose to whichpolyalbumin is covalently bound.

These remarkably higher yields are due to the more gentle elutingconditions made possible by employing the carrier of the invention. Inaddition, it can be seen that it is only the covalent binding of HSA tothe carrier that results in the inventive characteristic of thepre-S(2)-affinity and that a molecular sieve to which albumin is boundmerely by adsorption is not suitable for affinity chromatographicpurification.

What we claim is:
 1. A complex comprised of (i) an insoluble polymercarrier, (ii) monomeric human albumin and (iii) a pre-S hepatitis Bsurface antigen comprising a pre-S(2)-region, wherein said monomerichuman albumin is covalently bound to said carrier and said pre-Shepatitis B surface antigen is bound in an elutable form to saidmonomeric human albumin by said pre-S(2)-region.
 2. A complex as setforth in claim 1, wherein said insoluble polymer carrier is based on asubstance selected from the group consisting of agarose and dextrane. 3.A method of purifying pre-S hepatitis B surface antigen by using acomplex comprised of an insoluble polymer carrier, monomeric humanalbumin, and a pre-S hepatitis B surface antigen comprising apre-S(2)-region, wherein said monomeric human albumin is covalentlybound to said carrier and said pre-S hepatitis B surface antigen isbound in an elutable form to said monomeric human albumin by saidpre-S(2)-region, which method comprises the steps of:washing saidcomplex with a buffer solution to remove possibly present impurities andeluting said complex and recovering said pre-S hepatitis B surfaceantigen.
 4. A method as set forth in claim 3, wherein said pre-Shepatitis B surface antigen is recovered by treating said complex withan eluting agent containing chaotropic substances.
 5. A method as setforth in claim 4, wherein said chaotropic substances are selected fromthe group consisting of urea, guanuidine hydrochloride, thiocyanate,potassium chloride, magnesium chloride and potassium iodide.
 6. A methodas set forth in claim 3, wherein said pre-S hepatitis B surface antigenis recovered by treating said complex with a detergent.
 7. A method asset forth in claim 3, wherein said pre-S hepatitis B surface antigen isrecovered by treating said complex with a pH modifying agent.
 8. Amethod as set forth in claim 3, further comprising a secondarypurification step.
 9. A method as set forth in claim 6, wherein saiddetergent is selected from the group consisting of an ionic detergent, azwitterionic detergent and a non-ionic detergent.
 10. A method as setforth in claim 9, wherein said ionic detergent is selected from thegroup consisting of sodium desoxycholate and taurodesoxycholic acid. 11.A method as set forth in claim 9, wherein said zwitterionic detergent isselected form the group consisting of3[(3-cholamidopropyl)-dimethyl-ammonio]-1-propane sulfonate and3[(3-cholamidopropyl)-dimethyl-ammonio]-2-hydroxy-1-propane sulfonate.12. A method as set forth in claim 9, wherein said non-ionic detergentis octyl glucopyranoside.
 13. A method as set forth in claim 8, whereinsaid secondary purification step comprises a process selected from thegroup consisting of gel filtration, ultracentrifugation, hydrophobicchromatography, lectin affinity chromatography and ion exchangechromatography.